Fig 1: MIC-1 detection and quantitation in human prostate tissues (commercial tissue microarray).(A-B) Immunofluorescence with anti-MIC-1 antibody in a representative prostate tumor (A) and tumor adjacent tissue (B); pictures represent overlays of nuclear staining by DAPI (blue) and Alexa Fluor 488 immunostaining (yellow/white); the insets are Alexa Fluor 488 immunostaining only; white bars represent 10 micrometers. The diamond, closed arrow, and open arrow in B denote a typical lumen, epithelial cell compartment, and stromal cell compartment, respectively. (C-D) MIC-1 expression levels (indicated as signal intensities [pixel count]) in matched tumor adjacent and tumor tissues; the types of analysis were the following (as per Materials and Methods): (C) Whole slide analysis (WSA), (D) region of interest (ROI) analysis. Individual data points are shown as small black squares (partially overlapping); the boxes represent group medians (line across middle) and quartiles (25th and 75th percentiles) at its ends; lines above and below boxes indicate 10th and 90th percentiles, respectively. For each analysis, the number of images and cases is indicated; p values above the panels denote the level of statistical significance for the differences between groups, as calculated by the student’s t-test (p(t)) and by the Wilcoxon rank sums test (p(WRS)).
Fig 2: Detection of intracellular and mitochondrial levels of reactive oxygen species (ROS) in SH-SY5Y cells. (A) MitoSOX/MitoTracker/Hoechst staining images and fluorescence microscopy were used to determine the level of mitochondrial ROS in rotenone-treated SH-SY5Y cells. Hoechst (blue), Mito-Sox (red), and MitoTracker (green). The ROS level was significantly increased in cells not overexpressing GDF15 compared with that in normal control and GDF15-overexpressing cells. Magnification: 400×. (B) Quantification of mitochondrial ROS levels using flow cytometry and Mito-Sox-Red fluorescent probes. (C,D) Intracellular ROS generation (%) in SH-SY5Y cells was assessed with the ROS-sensitive fluorometric probe DCFH-DA using flow cytometry. Data are presented as the mean ± S.E. from three independent experiments, and differences were analyzed with an unpaired student t-test. **P < 0.01, compared to the control group; ##P < 0.01, as compared to rotenone-treated only group.
Fig 3: GDF15 decreases rotenone-induced SH-SY5Y cell apoptosis and prevents rotenone-induced mitochondrial damage. (A,B) Cell apoptosis was measured by flow cytometry after exposing cells to rotenone for 24 h. Flow cytometry showed an increased percentage of apoptotic cells after treatment with rotenone, whereas cells overexpressing GDF15 showed lower levels of apoptosis in comparison with those in cells only treated with rotenone. (C,D) A TUNEL assay was used to examine SH-SY5Y cell apoptosis. Rotenone led to severe SH-SY5Y cell apoptosis compared to that in control and GDF15-overexpressing cells. Data are presented as the mean ± S.E. from three independent experiments, and differences were analyzed with an unpaired student t-test. **P < 0.01, as compared to the control group; ##P < 0.01, as compared to rotenone-treated only group.
Fig 4: GDF15 expression is elevated in rotenone-treated SH-SY5Y cells and rotenone-induced cellular toxicity is reduced by GDF15. (A) The secretion of GDF15 protein was detected with an enzyme-linked immunosorbent assay after cells were exposed to rotenone. The level of GDF15 released showed a time-dependent increase, especially after 24 h. (B) Western blotting analysis showing that GDF15 expression was elevated and that tyrosine hydrolase (TH) was decreased in SH-SY5Y cells treated with rotenone for 24 and 36 h. (C) Cell viability was detected by a CCK8 assay in SH-SY5Y cells treated with 1 μM rotenone for 24 h and 0–100 ng/mL rhGDF15. (D–F) Western blotting analysis of GDF15, Bcl-2/Bax, p53, and PGC-1α in SH-SY5Y cells. β-Actin was used as the internal control. The overexpression of GDF15 had no evident effect on the expression of TH and Bcl-2/Bax under normal conditions. Compared to control group cells, p53 was found significantly increase with reduction of PGC-1α, Bcl-2/Bax and TH in rotenone treated group. And lower expression of p53 was detected in cells with GDF15 overexpression following rotenone treatment compared with rotenone treated-only groups, in addition to evident upregulation of the expression of Bcl-2/Bax, PGC-1α and TH. (G) GDF15 mRNA level determined using qRT-PCR. GDF15 mRNA level in GDF15 overexpression group was higher than that in normal control cell. And compared with rotenone only treated group, the GDF15 mRNA level was also found higher after rotenone treatment in GDF15overexpression cells. (H) mRNA expression normalized to that of GAPDH measured using PCR. GDF15 overexpression in SH-SY5Y cells had no evident effect on the expression of α-syn under normal conditions, but cell exposure to rotenone further increased the expression of α-syn and p53 and decreased the expression of Bcl-2/Bax and PGC-1α, whereas GDF15 overexpression could efficiently alleviate these changes. Data represent the mean ± S.E. from three independent experiments, and differences were analyzed with an unpaired student t-test. *P < 0.05, **P < 0.01 as compared to the control group; #P < 0.05, ##P < 0.01, as compared to rotenone-treated only group.
Fig 5: Expression patterns of conserved irradiation response genes in BMSCs. Venn diagrams for up- (a) and downregulated (b) genes shared by the P6 and P10 BMSCs according to the DEG analysis. c The expression pattern of the upregulated genes: each cluster of genes was distinguished accordingly by colour. d Expression patterns of the downregulated genes: each cluster of genes was distinguished accordingly by colour. e Expression pattern of genes that were either upregulated or downregulated at both 8 h and 2 h after irradiation relative to 0 h. f Relative expression levels of GDF15, CDKN1A and MDM2 measured by RNA-seq (FPKM) (up) and qRT-PCR (down). For qPCR, actin was used as the reference gene, and non-irradiated P6 BMSCs were used as the control groups. g Western blotting to evaluate CDKN1A, GDF15, HUJRP and p53 expression. All western blots are representative of three independent experiments. h Representative immunofluorescence staining in BMSCs. DAPI (blue), HJURP (red), merged images and quantification of immunofluorescence intensity (right) were shown. The photos were selected randomly. Scale bar 50 μm. Data are represented as the mean ± SEM. Student’s t test was performed to compare P6 and P10 BMSCs with significance set at a P value of less than 0.05. *P < 0.05, **P < 0.01. The same letter (lowercase for P10 and uppercase for P6, respectively) indicates no significant difference among different post-irradiation time (Tukey HSD, P < 0.05)
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